Acoustic cell patterning technologies represent an exciting potential tool to advance neuroscience research and neuroregenerative therapies. This technology relies on the use of ultrasound standing wave fields (USWFs) to rapidly and noninvasively pattern cells within 3D hydrogels. Ultrasound fields are highly controllable, allowing design of optimized exposure scenarios. This high risk, high reward proposal outlines proof-of-concept studies for USWF-mediated neural cell patterning, the results of which will provide support for the broader development and adoption of acoustic cell patterning technologies for neural tissue engineering applications. The overall goal of this project is to build and test the functionality of USWF exposure systems to spatially pattern neural cells within 3D hydrogels, and to identify acoustic patterning parameters that promote cell viability, control neural network morphology, and enhance neuronal activity. The project is comprised of three specific aims: i) Establish procedures to acoustically pattern neural cells in 3D collagen hydrogels, and identify USWF-patterning parameters that lead to increased neuronal cell viability and function; ii) Test different hydrogel formulations to determine whether addition of soluble recombinant fibronectin matrix analogs or co-patterning of endothelial and neural cells increases the neuroinductive capacity of USWF-fabricated hydrogels; and iii) Test the feasibility of translating USWF-mediated neural cell patterning technologies to neural tissue engineering applications by fabricating neural constructs, in vitro and in situ. Successful completion of this project will demonstrate the utility of this ultrasound technology for fabricating artificial 3D neural constructs for use as microphysiological systems, and demonstrate feasibility of translating the technology for neural guidance and regeneration in vivo. As such, this project has the potential to provide the neuroscience community with a novel cell patterning technology that can volumetrically organize neurons and glial cells alone, and in combination, to rapidly produce functional neural constructs.